This invention relates to arrays, particularly biopolymer arrays such as DNA or protein arrays, which are useful in diagnostic, screening, gene expression analysis, and other applications.
Polynucleotide arrays (such as DNA or RNA arrays) and peptide array, are known and may be used, for example, as diagnostic or screening tools. Such arrays include regions (sometimes referenced as spots or features) of usually different sequence polynucleotides or peptides arranged in a predetermined configuration on a substrate. The array is xe2x80x9caddressablexe2x80x9d in that different features have different predetermined locations (xe2x80x9caddressesxe2x80x9d) on a substrate carrying the array.
Biopolymer arrays can be fabricated using in situ synthesis methods or deposition of the previously obtained biopolymers. The in situ fabrication methods include those described in U.S. Pat. No. 5,449,754 for synthesizing peptide arrays, and in U.S. Pat. No. 6,180,351 and WO 98/41531 and the references cited therein for polynucleotides. In situ methods also include photolithographic techniques such as described, for example, in WO 91/07087, WO 92/10587, WO 92/10588, and U.S. Pat. No. 5,143,854. The deposition methods basically involve depositing biopolymers at predetermined locations on a substrate which are suitably activated such that the biopolymers can link thereto. Biopolymers of different sequence may be deposited at different feature locations on the substrate to yield the completed array. Procedures known in the art for deposition of biopolymers, particularly DNA such as whole oligomers or cDNA, are described, for example, in U.S. Pat. No. 5,807,522 (touching drop dispensers to a substrate), and in PCT publications WO 95/25116 and WO 98/41531, and elsewhere (use of a pulse jet in the form of a piezoelectric inkjet head).
Further details of large scale fabrication of biopolymer arrays by depositing either previously obtained biopolymers or by the in situ method, are disclosed in U.S. Pat. No. 6,242,266, U.S. Pat. No. 6,232,072, U.S. Pat. No. 6,180,351, and U.S. Pat. No. 6,171,797.
In array fabrication, the quantities of DNA available for the array are usually very small and expensive. Sample quantities available for testing are usually also very small and it is therefore desirable to simultaneously test the same sample against a large number of different probes on an array. These conditions require the manufacture and use of arrays with large numbers of very small, closely spaced features.
The arrays, when exposed to a sample, will exhibit a binding pattern. The array can be read by observing this binding pattern by, for example, labeling all targets such as polynucleotide targets (for example, DNA), in the sample with a suitable label (such as a fluorescent compound), scanning an illuminating beam across the array and accurately detecting the fluorescent signal from the different features of the array. Assuming that the different sequence polynucleotides were correctly deposited in accordance with the predetermined configuration, then the observed binding pattern will be indicative of the presence and/or concentration of one or more polynucleotide components in the sample. Peptide or arrays of other chemical moieties can be used in a similar manner. Conventionally, the illuminating and detecting have been performed on a dry array from a forward direction facing a front surface of the array carrying the array features, so that the illuminating and detected light need not pass through the substrate. In an alternative known arrangement, a transparent substrate forms part of a chamber in a housing with the array on a front substrate surface facing inward to the chamber. After exposure to a liquid containing the sample, the chamber is flushed and again filled with a liquid and the liquid containing chamber positioned in the array reading apparatus. In this situation, aside from the flushing and re-filling of the chamber, care must be taken that liquid does not leak from the chamber while positioned in the reading apparatus. The illuminating and detecting in this case has, of necessity, been performed in a backward direction through the substrate and onto the array while it is immersed in the liquid.
Techniques and apparatus for scanning chemical arrays are described, for example, in U.S. Pat. No. 5,763,870 and U.S. Pat. No. 5,945,679. Apparatus which reads an array by scanning an illuminating beam by the foregoing technique are often referred to as scanners and the technique itself often referred to as scanning. Conventionally, such scanning has been done by illuminating array features on a front surface of the substrate one pixel at a time.
Array scanners typically use a laser beam as a light source, which is scanned over pixels covering the array features. A detector (typically a fluorescence detector) with a very high light sensitivity is normally desirable to achieve maximum signal-to-noise in detecting hybridized molecules, particularly in array scanners used for DNA sequencing or gene expression studies. At present, photomultiplier tubes (xe2x80x9cPMTsxe2x80x9d) are still the detector of choice although charge coupled devices (xe2x80x9cCCDsxe2x80x9d) and avalanche photodiodes (xe2x80x9cAPDsxe2x80x9d) can also be used. PMTs and APDs are typically used for temporally sequential scanning of array features, while CCDs permit scanning many features in parallel (for example, one line of features simultaneously, in which case an illuminating line may be used).
When a sample component only weakly binds to an array feature (due to a low concentration of that component in the sample) the resulting fluorescence signal from that feature will be low. To be able to detect such low signal features, it is important to detect the resulting low signal with a high signal to noise ratio. It is also desirable to have a reading method where a liquid filled chamber containing the array is not positioned within the scanner.
One of the items recognized by the present invention is that when a dry array on a front surface of a substrate is interrogated or read from a forward position, the power density of the interrogating light or light emitted in response to the interrogating light, or both, can be reduced as a result of Fresnel reflection. This may consequently lead to a reduced detected signal from an array feature.
In one aspect then, the present invention provides a method of interrogating an addressable array unit having a substantially unreflective substrate with a front surface, and a dry array on the front surface having a plurality of different chemical features (for example, polynucleotide features of different sequence). In one configuration the method may include directing an interrogating light from a position forward of the front surface onto the chemical features of the dry array. In an additional or alternative configuration the method may include detecting light directed in a forward direction which is emitted from respective features in response to the interrogating light. The substrate may be substantially unreflective by virtue of there being no metallic or semi-metallic (for example, no silicon) coating present, and can also optionally be a transparent substrate.
In a first configuration, the detected light is emitted from locations of the features positioned within an xe2x85x9 wavelength of an antinode resulting from either directly incident interrogating light (that is, directly incident on the emitting locations) and interrogating light reflected by the substrate (to the emitting locations), or from directly detected emitted light and emitted light reflected by the substrate. That is, the majority (or optionally at least 60%, 70%, 80%, 90% or 95%) of detected light is from such features. In a second configuration the substantially unreflective substrate may include a transparent layer extending between the front surface of the substrate and a next layer, and which transparent layer has a thickness t. This transparent layer may function as a spacer layer or anti-reflective layer, as discussed below, and the thickness t may be any of those values used for distance d discussed below. In a third configuration (which may be in conjunction or not with the first or second configurations), the detected light power density for each of multiple features is within 30% (or 25, 20, 15, or 10%) of a maximum attainable under the same conditions with only the distance of the emitting locations from the front surface being varied, or the detected light power density is at least 10% (or 25, 33, or 50%) greater than attained under the same conditions with the anti-reflection layer absent (in which case the features are attached directly to a front surface of a base layer). By xe2x80x9csame conditionsxe2x80x9d in this context includes the same array/substrate and same instrument (and therefore with the same interrogating light illumination and emitted light detection, as well as the same depth of field for the detected emitted light, and with the focal plane adjusted to the same position relative to the front surface of the substrate).
The detected light may be emitted from locations which are spaced from the front surface of the substrate, or from an interface of two layers of the substrate having different refractive indices, by a distance d wherein:
nxcex/8 less than d less than (n+2)xcex/8 
where n is one or any second odd integer thereafter (that is, a member of the series 1, 5, 9, 13, 17 and so on), and xcex is the wavelength of the interrogating light or emitted light in the media from which the layer is made. When the distance d is that from the front surface of the substantially unreflective substrate, one way to accomplish this is by providing features which extend from the front surface by the distance d. When the distance d is that from an interface of two layers of different refractive index, this can be accomplished by one of the layers being a transparent layer of thickness t equal to d as defined above, and which extends between the front surface and a next layer. An alternative way is providing such transparent layer with a thickness which together with the distance which the features extend from the front surface of the substrate, totals to distance d as defined above.
The present invention further provides an addressable array unit of a type used in a method of the present invention. Such an array unit may have a substantially unreflective substrate with a front surface, and a dry array on the front surface having a plurality of different chemical features. In such an array unit the features may extend from the front surface by d above, for example, at least 80 nm (or 100, 125 or 150 nm) to less than 250 nm (or 225, 200, or 17 nm). In another construction, the substrate may include an anti-reflection or spacer layer extending between front surface and a next layer which may have a thickness equal to or less than any of the foregoing thickness ranges. Since the array may not have been exposed to a sample at this point (for example, to a sample containing many fluorescently labeled polynucleotides of different sequence which hybridize to respective polynucleotide array features), this thickness may only provide an indication of the distance by which the light emitting features will be spaced from the front surface of the substrate during reading of the dry array. After exposure to a sample (such as a sample containing fluorescently labeled polynucleotides) the locations from which light is emitted (for example, the fluorescent labels) upon reading of the array may be spaced a distance d as defined above from the substrate front surface. The array unit may optionally include instructions to interrogate the array by a method of the present invention, and/or with one or more parameters described herein (for example, direct the interrogating light onto the front side of the substrate). An identifier carried on the array substrate, or a housing carrying the substrate, may provide such instructions.
In a method of the present invention, the interrogating light may be directed toward the front surface at an angle of less than 50 degrees to a normal to the back surface (such as less than 36, 25, 20. 15, or less than 5 degrees), and more than 0, 1, 2 or 4 degrees, as well as 0 degrees itself (where the interrogating light is perpendicular to the front surface). The same ranges may be used for the detected light leaving the back surface.
The present invention further provides a method for use with an addressable array unit having a transparent substrate with a front surface, and an array with a plurality of different chemical features on a front surface. The method includes reading (such as machine reading) an identifier associated with the array unit (such as by being present on the substrate carrying the array, a housing carrying the substrate, or in or on a same package with the array substrate). An instruction that the array should be interrogated and read from a forward direction (that is, forward of the front surface), is retrieved (such as by a processor) based on the read identifier. The instruction may be retrieved from the read identifier itself, or from a memory using data from the read identifier (for example, the whole or part of the retrieved identifier). The retrieved instruction may be used to check that the array is properly oriented within an array reader such that the array can be interrogated and read by the reader from the forward direction.
Another aspect of the present invention provides an apparatus for reading an array which illuminates the array and detects light emitted in response thereto, from the forward direction in a manner as already described. The apparatus includes a light source to provide the interrogating light, and a detector to receive the emitted light. A processor receives the data from the detector and may save the results (either further processed or raw) in a memory. The processor may also execute any other method of the present invention, such as retrieving the instruction based on the read identifier, and checking that the array is properly oriented such as based on signals received from the detector or another means (for example, based on whether an indicia, such as the identifier or other indicia, is facing in the correct direction corresponding to proper orientation of the array in the apparatus). The present invention further provides a computer program product for use with such a chemical array reader apparatus. The computer program product comprises a computer readable storage medium having a computer program stored thereon which, when loaded into the processor, causes the reader to execute a method as describe herein.
While the methods and apparatus have been described in connection with arrays of various moieties, such as polynucleotides (for example, DNA), it will be understood throughout this description that other moieties can be used and may include any chemical moieties such as other biopolymers or polymers.
The present invention can provide any one or more of the following or other benefits. For example, one or more advantages of reading an array from the front side (for example, relative insensitivity to typical substrate defects or thickness, back surface quality, and variations in refractive index) may be obtained while reducing deleterious signal loss due to Fresnel reflections.